Abstract
S-nitrosylation (SNO) is an important oxidative posttranslational modification in the regulation of cardiac mitochondria. SNO modification of several mitochondrial proteins has been associated with cardiac preconditioning and improved cell survival following ischemia/reperfusion injury. Due to their labile nature, SNO modifications are challenging to study using traditional biochemical techniques; particularly, the identification of individual modified cysteine residues. Here, we describe the details of the cysTMT6 switch assay, a variation of the classic biotin switch protocol. The cysTMT6 reagent provides a simplified and powerful approach to SNO detection by combining unambiguous identification of the modified cysteine residue and relative quantification of up to six samples by mass spectrometry analysis.
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References
Jones SP, Bolli R (2006) The ubiquitous role of nitric oxide in cardioprotection. J Mol Cell Cardiol 40:16–23
Foster DB et al (2009) Redox signaling and protein phosphorylation in mitochondria: progress and prospects. J Bioenerg Biomembr 41:159–168
Borutaite V, Brown GC (2006) S-nitrosothiol inhibition of mitochondrial complex I causes a reversible increase in mitochondrial hydrogen peroxide production. Biochim Biophys Acta 1757:562–566
Chouchani ET et al (2010) Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation. Biochem J 430:49–59
Dahm CC et al (2006) Persistent S-nitrosation of complex I and other mitochondrial membrane proteins by S-nitrosothiols but not nitric oxide or peroxynitrite: implications for the interaction of nitric oxide with mitochondria. J Biol Chem 281:10056–10065
Burwell LS et al (2006) Direct evidence for S-nitrosation of mitochondrial complex I. Biochem J 394:627–634
Sun J et al (2007) Preconditioning results in S-nitrosylation of proteins involved in regulation of mitochondrial energetics and calcium transport. Circ Res 101:1155–1163
Burwell LS, Brookes PS (2008) Mitochondria as a target for the cardioprotective effects of nitric oxide in ischemia-reperfusion injury. Antioxid Redox Signal 10:579–599
Prime TA et al (2009) A mitochondria-targeted S-nitrosothiol modulates respiration, nitrosates thiols, and protects against ischemia-reperfusion injury. Proc Natl Acad Sci USA 106:10764–10769
Nadtochiy SM et al (2009) In vivo cardioprotection by S-nitroso-2-mercaptopropionyl glycine. J Mol Cell Cardiol 46:960–968
Halestrap AP et al (2007) The role of mitochondria in protection of the heart by preconditioning. Biochim Biophys Acta 1767:1007–1031
Murphy E, Steenbergen C (2007) Preconditioning: the mitochondrial connection. Annu Rev Physiol 69:51–67
Jaffrey SR et al (2001) Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat Cell Biol 3:193–197
Jaffrey SR, Snyder SH (2001) The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE, pl1
Hao G et al (2006) SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures. Proc Natl Acad Sci USA 103:1012–1017
Forrester MT et al (2009) Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture. Nat Biotechnol 27:557–559
Huang B, Chen C (2010) Detection of protein S-nitrosation using irreversible biotinylation procedures (IBP). Free Radic Biol Med 49:447–456
Paige JS et al (2008) Nitrosothiol reactivity profiling identifies S-nitrosylated proteins with unexpected stability. Chem Biol 15:1307–1316
Murray CI et al (2011) Identification and quantification of S-nitrosylation by cysteine reactive tandem mass tag switch assay. Mol Cell Proteomics 11:M111.013441
Zhou X et al (2010) ESNOQ, proteomic quantification of endogenous S-nitrosation. PLoS One 5:e10015
Cavadini P et al (2002) Protein import and processing reconstituted with isolated rat liver mitochondria and recombinant mitochondrial processing peptidase. Methods 26:298–306
Storrie B, Madden EA (1990) Isolation of subcellular organelles. Methods Enzymol 182:203–225
Abadir PM et al (2011) Identification and characterization of a functional mitochondrial angiotensin system. Proc Natl Acad Sci USA 108:14849–14854
Hong HY et al (2000) Direct Blue 71 staining of proteins bound to blotting membranes. Electrophoresis 21:841–845
Park JK, Kostka P (1997) Fluorometric detection of biological S-nitrosothiols. Anal Biochem 249:61–66
Kanai AJ et al (1997) Beta-adrenergic regulation of constitutive nitric oxide synthase in cardiac myocytes. Am J Physiol 273:C1371–C1377
Pinsky DJ et al (1997) Mechanical transduction of nitric oxide synthesis in the beating heart. Circ Res 81:372–379
Acknowledgement
The authors would like to thank John Rogers at Thermo Fisher Scientific for the generous gift of cysTMT materials. This work was supported by American Heart Association Pre-Doctoral Fellowship 0815145E to CIM and NIH grants P01 HL77180-0, N01-HV-28180, and P50 HL 084946-01 to JVE.
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Murray, C.I., Chung, H.S., Uhrigshardt, H., Van Eyk, J.E. (2013). Quantification of Mitochondrial S-Nitrosylation by CysTMT6 Switch Assay. In: Vivanco, F. (eds) Heart Proteomics. Methods in Molecular Biology, vol 1005. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-386-2_14
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DOI: https://doi.org/10.1007/978-1-62703-386-2_14
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